The objective of this research is to study mutagenesis at the DNA level in mammals and teleost fish. The fish will be used for research on ova and on differentiation. A major problem in mutagenesis is that the level and specificity of response are very different between indicator organisms; predictions abut induced mutagenesis may not be relevant. Significant variation is due to the diversity of marker genes; a single sequence needs to be used among various organisms and tissues. The use of integrated transgenic vector can combine a theoretical study of mutations in several model organisms with assessment of mutagenic hazard and development of biomonitoring systems for aquatic biospheres. In the initial experiments, the well-characterized am3 of phiX174 has been used to evaluate substitutions at the A:T base pair. Inbred C57B1/6 mice have been made homozygous for 100 copies of phiX174 in a tandem array; we have not observed any detrimental effect from the insertion. Reversions via one transition and two transversion are detected by selection among progeny phage recovered from the transgenic mice. Both the recovery and the putative mutation frequencies are independent of any endogenous variation in CpG methylation among host animals or tissues. Spontaneous mutation frequencies are in the range of 2-4x10,000,000 for liver, spleen, brain, kidney and testis. In adult animals treated with 200 mg/kg N-methyl-N- nitrosourea (ENU), there are significant increases in mutation frequencies in the spleen and liver, but not the kidney or brain. The accomplishments for the fish studies are: 1) Produced several transgenic founders of Fundulus and Medaka containing phiX174 as a single gene marker and 2) Demonstrated that the transgenic vector can be efficiently recovered from the chromosomal DNA of fish. We are in the process of establishing the spontaneous mutation frequency for one line of Fundulus. Efforts are underway to produce additional transgenic mice containing other specific markers for analysis of CpG-dependent and CpG-independent mutagenesis at the single gene copy level. The use of well-defined phage may allow us to compare spontaneous and induced intragenic events, recombination or developmental mosaicism in both somatic and gametogenic tissue with an overlay of comparison between distant related vertebrate.